The broad, long-term objectives of our laboratory are to define unique regions of structure and function in viral RNAs, allowing them to be attacked without sustaining heavy cellular damage. There are millions of patients with chronic HCV infection in the United States and abroad, but the efficiency of present therapies is a difficult problem. HCV is thus a leading candidate for new antiviral therapies based on targeting unique RNA sequences, structures and functions of the virus. Since such treatments will work best in conserved sequences of the HCV genome, our project will focus on the 5' terminal domain of the viral RNA, which includes the Internal Ribosome Entry Site (IRES), the unusual structure with which HCV and several other RNA viruses signal protein synthesis initiation, in striking contrast to cellular mRNA molecules, which employ a conventional 7-methyl guanosine cap structure. The specific aims of this proposal are: (i) To map the conserved 5' terminal 400 bases of the HCV genome for regions of conserved sequence, secondary structure, and local tertiary structural elements, emphasizing unique regions combining all three elements. Techniques we already use, including oligonucleotide binding, gel shift, RNase H-directed cleavage, RNase III digestion and RNA-RNA crosslinking by UV light will all be employed. (ii) We will employ ribosome binding and protein synthesis assays to identify the exact RNA domains needed for IRES function, and will test modified and mutant sequences to map their fine structure. Our direct ribosome binding site isolation method employing RNAase protection in the reticulocyte lysate will be employed to pinpoint these IRES sequences and their structures derived in aim (i). (iii) We will design ribozyme targeting sequences and other agents which will be directed against key functional domains characterized in aims (i) and (ii). Included will be design and testing of External Guide Sequences (EGSs) for human RNase P in liver cells.